Several clinical laboratories assess sperm DNA fragmentation (sDF) in addition to semen analysis in male infertility diagnosis. Among tests evaluating sDF, TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) and SCD (Sperm Chromatin Dispersion) are widely used. Our lab developed a modified version of TUNEL (TUNEL/PI) able to distinguish two sperm populations (PI Brighter and PI Dimmer) differently associated with sperm viability and reproductive outcomes. The aim of this study was to compare sDF levels detected by SCD and TUNEL/PI in the semen samples from 71 male subjects attending our Andrology Laboratory. Our results demonstrate that SCD is less sensitive in determining sDF compared to TUNEL/PI. The statistically significant positive correlation found between sDF evaluated by SCD and PI Dimmer (consisting of all dead spermatozoa) suggests that SCD mainly detects sDF in unviable spermatozoa. We confirmed that most spermatozoa detected by SCD are unviable by performing SCD after incubation in hypo-osmotic medium to discriminate viable and unviable cells in 52 samples. Such results might explain the lower ability of this test in discriminating couples having successful ART outcomes demonstrated in published metanalyses. Overall, our results indicate that SCD is less sensitive in evaluating sDF for diagnostic purposes.

Many viruses infect the male genital tract with harmful consequences at individual and population levels. In fact, viral infections may induce damage to different organs of the male genital tract (MGT), therefore compromising male fertility. The oxidative stress, induced during viral-mediated local and systemic inflammation, is responsible for testicular damage, compromising germinal and endocrine cell functions. A reduction in sperm count, motility, number of normal sperm and an increase in DNA fragmentation are all common findings in the course of viral infections that, however, generally regress after infection clearance. In some cases, however, viral shedding persists for a long time leading to unexpected sexual transmission, even after the disappearance of the viral load from the blood.The recent outbreak of Zika and Ebola Virus evidenced how the MGT could represent a reservoir of dangerous emergent viruses and how new modalities of surveillance of survivors are strongly needed to limit viral transmission among the general population.Here we reviewed the evidence concerning the presence of relevant viruses, including emergent and re-emergent, on the male genital tract, their route of entry, their adverse effects on male fertility and the pattern of viral shedding in the semen.We also described laboratory strategies to reduce the risk of horizontal or vertical cross-infection in serodiscordant couples undergoing assisted reproductive technologies.

Background: Sperm cryopreservation is a consolidate option for long‐term male fertility preservation. The freezing/thawing procedure causes detrimental effects to spermatozoa, including damage to viability, motility, membrane composition, and DNA, whereas the effect on sperm chromatin compaction is less studied. Objectives: The primary aim of this study was to investigate the impact of cryopreservation on sperm chromatin compaction. Furthermore, the effect of cryopreservation on sperm parameters (motility, viability, chromatin compaction, and DNA fragmentation) was also assessed in relation to the storage time in liquid nitrogen. Materials and Methods: Semen samples, collected from 126 (92 normozoospermic and 34 oligozoospermic) patients undergoing routine semen analysis in the Andrology Laboratory of Careggi University Hospital of Florence, were frozen by conventional fast vapor freezing method. Sperm motility, viability, kinematic parameters (by computer‐aided sperm analysis [CASA]), chromatin compaction (by staining with both aniline blue [AB] and Chromomycin A3 [CMA3]), and sperm DNA fragmentation (sDF, by TUNEL/Propidium Iodide [PI]) were evaluated before freezing and after thawing at different timepoints. Results: After 7 days of storage, a significant decline in sperm motility, viability, and kinematics parameters, as well as a significant increase in the percentage of sperm positivity to CMA3, AB, and sDF, were observed. It is noteworthy that while motility and viability decreased in almost all subjects, the increase in CMA3 and AB positivity was observed in 68.0% and 79.2% of samples, respectively. A progressive deterioration of sperm motility and viability, less evident for chromatin structure, was observed at longer times of storage (28 and 180 days). Discussion: Our results indicate that freezing/thawing procedures can alter chromatin structure. A reduction in protamine content and/or a modification in chromatin assembly can be hypothesized. Furthermore, the length of storage in liquid nitrogen appears to progressively affect sperm parameters, although it should be confirmed in larger cohort of subjects. Conclusion: Current sperm cryopreservation protocols need to be improved with new strategies and personalized procedures aimed to minimize the damage.

Background: Sperm cryopreservation is recommended to preserve male fertility for cancer patients or other medical conditions at risk of sperm decline. Whether motility and viability recovery rates vary depending on the medical conditions requiring cryopreservation is poorly known. We report here on the 24-year experience of our semen bank. Methods: Motility and viability recovery rates were evaluated in 1973 collections from patients with various medical conditions and 67 collections from donors, and the results were related to basal semen quality. Results: Motility and viability recovery were highly related to basal semen quality and varied between cancer and non-cancer conditions, independently of the duration of cryopreservation and patient age. In samples with a sperm number below 2 × 10⁶/mL, recovery rates approximated to zero. The highest recovery rates were found in donor collections. Cut-off values for the recovery of at least 1% motile spermatozoa were established based on initial semen quality. Conclusions: Our results indicate that the occurrence of any pathological or medical condition resulted in lower recovery rates with respect to donors, indicating that intrinsic sperm characteristics drive susceptibility to cryodamage. Established cut-off values for motility recovery can be useful for patient counseling as well as for ART laboratories to decide the type of procedure.

Background: Sperm cryopreservation is an important procedure for oligozoospermic subjects at risk of azoospermia and after surgical recovery of spermatozoa in non‐obstructive azoospermic men. Conventional procedures for sperm cryopreservation might be, however, not suitable for samples with a very low sperm number.Objectives: In this pilot study, we investigated the recoveries of sperm motility and viability in severe oligozoospermic subjects (n = 39) after cryopreservation with a tip‐microVapour Fast Freezing, a procedure previously developed by our group for men with good semen quality. Sperm DNA fragmentation was also evaluated in a second group of oligozoospermic samples (n = 16).Materials and methods: We used a Vapour Fast Freezing procedure using 10 μL tips as carrier, and Test Yolk Buffer as freezing medium (tip‐microVapour Fast Freezing). In a subset of samples (n = 22), we compared recovery of motility and viability as obtained with tip‐microVapour Fast Freezing and with a Vapour Fast Freezing procedure using 500 μL straws. Sperm DNA fragmentation was evaluated by the sperm chromatin dispersion test.Results: We found a recovery rate (median [interquartile range]) of 0.29 (0.13–0.41) for progressive motility, 0.30 (0.21–0.52) for total motility and 0.48 (0.29–0.60) for viability. Interestingly, we observed that samples with the poorest motility were apparently less damaged by freezing/thawing. In a subset of samples (n = 22), we directly compared values of viability, progressive motility and total motility by freezing/thawing with tip‐microVapour Fast Freezing and Vapour Fast Freezing conducted with 500 μL straws. We found much better values of all sperm parameters in samples after freezing/thawing with tip‐microVapour Fast Freezing than with Vapour Fast Freezing in 500 μL straws: that is, progressive motility: 7.00 (3.00–8.50)% versus 2.00 (0.00–4.25)%, p < 0.001; total motility: 12.00 (8.00–16.25)% versus 6.50 (1.00–9.25)%, p < 0.001; viability: 29.75 (23.75–45.25) versus 22.50 (13.75–28.13), p < 0.001, respectively. In the second group of oligozoospermic samples, we found that tip‐microVapour Fast Freezing produced lower levels of sperm DNA fragmentation than straws (33.00 [19.75–36.00]% vs. 36.00 [22.75–41.87]%, p < 0.001).Discussion and conclusion: Tip‐microVapour Fast Freezing appears to be a very promising method to cryopreserve semen samples from severe oligozoospermic patients.